Magnetic resonance imaging agents for calcification

ABSTRACT

The present invention discloses magnetic resonance compatible contrast agents for water-poor structures, such as bone and tissue calcification. In particular, the present invention discloses bisphosphonate-based magnetic resonance imaging contrast agents specific for hydroxyapatite, the calcium salt most commonly associated with malignant calcification.

FIELD OF THE INVENTION

The present invention discloses magnetic resonance (MR)-compatiblecontrast agents for detection of water-poor structures, such as bone andtissue calcification.

BACKGROUND

Magnetic resonance imaging (MRI) has become one of the most widely usedimaging modalities in clinical practice that provides soft tissue imagesdepicting both anatomy and pathologies. Since MRI signal arises fromprotons, water-poor structures, such as bone and tissue calcification,are essentially invisible.

Tissue calcification is an important biomarker for human disease, withmicrocalcifications being of paramount importance for the detection ofbreast cancer. However, MRI, now the standard of care for screeninghigh-risk women for breast cancer, is unable to detect suchcalcifications.

About 80% of MRI protocols in North America employ injected contrastagents that improve tissue contrast and may give additional information{Caravan, 2006; Caravan, 1999}. The most commonly used MRI contrastagents are thermodynamic and kinetically stable low molecular weightgadolinium chelates that alter the relaxivity properties of thesurrounding water {Bottrill, 2006}. While a wide range of nonspecificcontrast agents are being used in clinical applications for evaluationof physiological parameters, the development of efficient targeted MRIcontrast agents directed at specific molecular entities has dramaticallyexpanded the range of possible applications for MRI by combining thenoninvasiveness and high spatial resolution of MRI with the specificlocalization of molecular targets {Weinmann, 2003}. However, previousstudies aimed at the development of bone-seeking agents, have shown thatthe Gd³⁺ complex of DOTP⁵⁻[1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonate)]failed to enhance the surrounding water signal when complexed to thebone {Alves, 2003}.

SUMMARY

Bisphosphonates (BPs) bind avidly to hydroxyapatite (HA) bone mineralsurfaces {van Beek, 1998} and have both diagnostic {Ogawa, 2005; Lam,2007} and therapeutic uses {Lipton, 2000}. BPs are analogues ofendogenous pyrophosphates in which the hydrolysable oxygen atom thatseparates the two phosphate groups is replaced with a more stable carbonatom. The P-C-P structure is responsible for giving BPs their highaffinity for bone, which can be further enhanced by addition of ahydroxyl group at the central carbon atom {van Beek, 1998}.

Osteoblastic bone lesions are typically diagnosed using BP-basedradiotracers {Lam, 2007}. MRI of bone lesions could provide superioranatomical localization, would eliminate ionizing radiation, and couldbe used to guide magnetic resonance spectroscopic evaluation.

The reason for the inapplicability of MRI to bone and/or solid andsemi-solid like structures is two-fold: (i) lack of free water in thesestructures, (ii) water that is present is partially bound, resulting inthe short transverse relaxation times (T₂). Thus, it is prohibitivelydifficult to visualize bone surfaces using conventional magneticresonance (MR) methodology. Recently, MR techniques employing variousultra short echo (UTE) signal acquisition schemes have become available{Irarrazabal, 1995; Song, 1998}. Rationale for exploiting UTE pulsesequence for MRI in present invention arises from previous work showingthat TE on the order of 100 μsec is capable of providing excellent MRIof bone {Bydder, 2006}. In such “solid-like” environments, transverserelaxation times (T₂) are very short, averaging≈1 msec for bone andseveral msec for periosteum. Because of these short T₂s, thesestructures are poorly seen using conventional gradient echo (GRE) orspin-echo sequences. However, UTE sequence alone is necessary but notsufficient for detecting calcification. Gd-based contrast agentsspecific for the calcium salt of interest, needed to be employed inconjunction with the UTE pulse sequence.

The present invention describes a preparation and application ofBP-based MRI contrast agent for UTE MRI detection of HAmicrocalcification, a hallmark of malignant breast cancer.

Complicating the development of BP-based MRI contrast agents, however,is the proclivity of BP's to bind lanthanides, the water-poorenvironment of the bone surface, and the difficulty of chemicalsynthesis. Unlike most contrast agents and radiotracers, which arerelatively immune to their aqueous environment, Gd-based MR contrastagents are highly sensitive to water (i.e., proton) access.

One aspect of the present invention seeks to provide BP-based MRIcontrast agents with different spacer length between derivatives of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and theBP. Because of propensity of BPs to chelate metals themselves {Alves,2003}, the BP can be added in a pre-loading strategy after metalchelation by DOTA (FIG. 1 and FIG. 2). Pre-loading of metals on DOTAeliminates the possibility of competition from in-vivo calcium for thephosphonate which could result in the release of toxic metals, such as,gadolinium. In such an aspect, linker, H₂N-A-COOH is an amino acid or Ais independently selected from an alkane, polyethylene glycol andpolypropylene glycol. M is Y, In, Gd, Eu, or a lanthanide. In oneembodiment, amino acid is natural amino acid. In some embodiment, aminoacid is unnatural amino acid. In some embodiment an alkane is C1-C20straight chain carbon unit. In some embodiments, polyethylene glycol is6 to 20 ethylene glycol unit. In some embodiments, polypropylene glycolis 6 to 20 propylene glycol unit. In some embodiments Eu is loaded forPARACEST contrast agent. In some embodiments, Y is loaded forhyperpolarized MRI contrast agent.

In an another aspect, methyl ester protected BP can be generated beforemetal chelation on an organic chelating ligand (FIG. 3 and FIG. 4).Methyl ester protected BP deprotection, after metal loading on anorganic chelating ligand, results in contrast agent. In such an aspect,linker, H₂N-A-COOH is amino acid or A is independently selected from analkane, polyethylene glycol and polypropylene glycol. M is Y, In, Gd,Eu, or a lanthanide. In one embodiment, amino acid is natural aminoacid. In some embodiment, amino acid is unnatural amino acid. In someembodiment an alkane is C1-C20 straight chain carbon unit. In someembodiments, polyethylene glycol is 6 to 20 ethylene glycol unit. Insome embodiments, polypropylene glycol is 6 to 20 propylene glycol unit.

In an another aspect, BPs are conjugated to an organic chelating ligand(FIG. 5 and FIG. 6) followed by metal chelation on an organic chelatingligand, results in contrast agent. In such an aspect, linker, H₂N-A-COOHis an amino acid or A is independently selected from an alkane,polyethylene glycol and polypropylene glycol. M is Y, In, Gd, Eu, or alanthanide. In one embodiment, amino acid is natural amino acid. In someembodiments, amino acid is unnatural amino acid. In some embodiments, analkane is C1-C20 straight chain carbon unit. In some embodiments,polyethylene glycol is 6 to 20 ethylene glycol unit. In someembodiments, polypropylene glycol is 6 to 20 propylene glycol unit.

In an another aspect, the present invention provides a contrast agentrepresented in general formula [I], and pharmaceutically acceptablesalts, hydrates and solvents thereof:

In such an aspect, BP is a bisphosphonate,

is a linker, and

is a metal chelate selected independently from:

In one embodiment, bisphosphonate is independently selected fromalendronate, etidronate, ibandronate, incadronate, neridronate,olpadronate, phosphonate, pamidronate, risedronate, tiludronate andzoledronate. In some embodiments, linker is independently selected fromamino acid, alkane, polyethylene glycol and polypropylene glycol. Insome embodiments, M is Y, In, Gd, Eu, or a lanthanide. In someembodiments Eu is loaded for PARACEST contrast agent. In someembodiments, Y is loaded for hyperpolarized MRI contrast agent. In someembodiments, amino acid is natural amino acid. In some embodiments,amino acid is unnatural amino acid. In some embodiments, an alkane isC1-C20 straight chain carbon unit. In some embodiments, polyethyleneglycol is 6 to 20 ethylene glycol unit. In some embodiments,polypropylene glycol is 6 to 20 propylene glycol unit.

The major medical application of present invention is in the highsensitivity MRI detection of tissue calcification, especiallymicrocalcification in breast cancer, without the need for ionizingradiation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 represent metal hepta coordinated BP-based MRI contrast agents inwhich BPs can be added in a pre-loaded strategy after metal chelation byDOTA.

FIG. 2 represent metal octa coordinated BP-based MRI contrast agents inwhich BPs can be added in a pre-loaded strategy after metal chelation byDOTA.

FIG. 3 represent metal hepta coordinated BP-based MRI contrast agents inwhich methylester protected BPs can be generated before metal chelationon an organic chelating ligand.

FIG. 4 represent metal octa coordinated BP-based MRI contrast agents inwhich methylester protected BPs can be generated before metal chelationon an organic chelating ligand.

FIG. 5 represent metal hepta coordinated BP-based MRI contrast agents inwhich BPs are conjugated to an organic chelating ligand followed bymetal loading on an organic chelating ligand.

FIG. 6 represent metal octa coordinated BP-based MRI contrast agents inwhich BPs are conjugated to an organic chelating ligand followed bymetal loading on an organic chelating ligand.

FIG. 7 is a synthetic scheme for preparation of [Gd³⁺-DOTA]-Thr-Pam-Na(Scheme 1).

FIG. 8 is a synthetic scheme for preparation of[Gd⁴⁺-DOTA]-(PEG)₈-Pam-Na (Scheme 2).

FIG. 9 is an alternative synthetic scheme for preparation of[Gd³⁺-DOTA]-Thr-Pam-Me (Scheme 3).

FIG. 10 is an alternative synthetic scheme for preparation of[Gd⁴⁺-DOTA]-(PEG)₈-Pam-Me (Scheme 4).

FIG. 11 is an alternative synthetic scheme for preparation of[Gd³⁺-DOTA]-Thr-Pam-Na (Scheme 5).

FIG. 12 is an alternative synthetic scheme for preparation of[Gd⁴⁺-DOTA]-(PEG)₈-Pam-Na (Scheme 6).

DETAILED DESCRIPTION

In a present invention, a synthetic strategy is developed for BP-basedMRI contrast agents particularly for water-poor structure such as bonelesions and tissue calcification, and more particularly for breastcancer microcalcification. BP-based MRI contrast agents are designed inwhich the small molecule BPs, a targeting ligand is engineered tocontain a primary amine for conjugation, and is optimized for bindingaffinity and physicochemical properties independent of the desiredfunctional molecules. Functional molecules are conjugated covalently tothe targeting ligands with linkers that provide adequate isolation ofthe two functions.

The BP-based MRI contrast agents of present invention are preparedaccording to the methods known in the art, as illustrated in general inFIGS. 1-6 and described for specific compounds in examples 1-6. Productsare characterized by analytical HPLC, NMR and LCMS, and are obtained intypical yields of 50-60%.

FIG. 1 of present invention describe a synthetic scheme for metal heptacoordinated BP-based MRI contrast agents in which BPs can be added in apre-loaded strategy after metal chelation by DOTA. Linker with terminalprimary amine and carboxylic acid functionality is conjugated withDOTA(tBu)₃-NHS and subsequent removal of protecting groups on carboxylicmoiety results in inermediate for metal chelation. Metal chelation isperformed by reaction with metal chloride. Carboxylic acid functionalgroup on DOTA pre-loaded with metal is activated and conjugated withprimary amine functional group of BP to results in BP-based MRI contrastagents.

In one aspect of present invention, a method for synthesizing a BP-basedMRI contrast agent is provided. The method involves steps of:

-   (a) Starting synthesis with an organic chelating ligand selected    from the group of:

where in one embodiment R is t-butyl ester, ester or hydrogen, and

-   R¹ is

-   (b) reacting an organic chelating ligand with a linker having a    primary amine and a carboxylic moiety at opposing ends, (c) treating    the carboxylic moiety with oxalyl chloride to form an acid chloride    at the carboxylic moiety, (d) reacting said acid chloride in one pot    with trialkyl phosphite and dialkyl phosphite to form a alkylester    protected BP, (e) deprotecting one or more carboxylic acid ester of    an organic chelating ligand to yield one or more carboxylic acid    functionality, (f) chelating a metal ion to result in a metal    chelate, where the linker separate the metal chelate and the    alkylester protected BP, and (g) deprotecting one or more BP ester    of the alkylester protected BP to results in the BP-based MRI    contrast agent.

In some embodiments, linker is independently selected from amino acid,alkane, polyethylene glycol and polypropylene glycol. In someembodiments, amino acid is natural amino acid. In some embodiments,amino acid is unnatural amino acid. In some embodiments, an alkane isC1-C20 straight chain carbon unit. In some embodiments, polyethyleneglycol is 6 to 20 ethylene glycol unit. In some embodiments,polypropylene glycol is 6 to 20 propylene glycol unit. In someembodiments, alkyl is methyl, ethyl or propyl. In some embodiments,metal ion is Y, In, Gd, Eu, or a lanthanide.

In an another aspect of present invention, a method for synthesizing aBP-based MRI contrast agent is provided. The method involves steps of:

-   (a) Starting synthesis with an organic chelating ligand selected    from the group of:

where in one embodiment R is t-butyl ester, ester or hydrogen, and

-   R¹ is

-   (b) reacting an organic chelating ligand with a linker having a    primary amine and a carboxylic moiety at opposing ends, (c)    deprotecting one or more carboxylic acid ester of the organic    chelating ligand to yield one or more carboxylic acid    functionality, (d) chelating a metal ion to result in a metal    chelate, where a metal chelate having a carboxylic moiety, and (e)    reacting an amino BP with carboxylic moiety of a metal chelate to    form an amide bond to results in BP-based MRI contrast agent.

In some embodiments, linker is independently selected from amino acid,alkane, polyethylene glycol and polypropylene glycol. In someembodiments, amino acid is natural amino acid. In some embodiments,amino acid is unnatural amino acid. In some embodiments, an alkane isC1-C20 straight chain carbon unit. In some embodiments, polyethyleneglycol is 6 to 20 ethylene glycol unit. In some embodiments,polypropylene glycol is 6 to 20 propylene glycol unit. In someembodiments, BP is independently selected from alendronate, neridronate,pamidronate, risedronate, tiludronate and zoledronate. In someembodiments, metal ion is Y, In, Gd, Eu, or a lanthanide.

In an another aspect of present invention, a method for synthesizing aBP-based MRI contrast agent is provided. The method involves steps of:

-   (a) Starting synthesis with an organic chelating ligand selected    from the group of:

where in one embodiment R is t-butyl ester, ester or hydrogen, and

-   R¹ is

-   (b) reacting an amino BP with an organic chelating ligand to form an    amide bond between a BP and an organic chelating ligand, (c)    deprotecting one or more carboxylic acid ester of an organic    chelating ligand to yield one or more carboxylic acid functionality,    and (d) chelating a metal ion to one or more carboxylic acid ester    of the organic chelating ligand to result in BP-based MRI contrast    agent.

In some embodiments, linker separates an organic chelating ligand andthe BP. In some embodiments, linker is independently selected from aminoacid, alkane, polyethylene glycol and polypropylene glycol. In someembodiments, amino acid is natural amino acid. In some embodiments,amino acid is unnatural amino acid. In some embodiments, an alkane isC1-C20 straight chain carbon unit. In some embodiments, polyethyleneglycol is 6 to 20 ethylene glycol unit. In some embodiments,polypropylene glycol is 6 to 20 propylene glycol unit. In someembodiments, BP is independently selected from alendronate, neridronate,pamidronate, risedronate, tiludronate and zoledronate. In someembodiments, metal ion is Y, In, Gd, Eu, or a lanthanide.

In an another aspect, the present invention provides a contrast agentrepresented in general formula [II], and pharmaceutically acceptablesalts, hydrates and solvents thereof:

In such an aspect, BP is a bisphosphonate,

is a linker, and

M is Y, In, Gd, Eu, or a lanthanide.

In one embodiment, bisphosphonate is independently selected fromalendronate, etidronate, ibandronate, incadronate, neridronate,olpadronate, phosphonate, pamidronate, risedronate, tiludronate andzoledronate. In some embodiments, linker is independently selected fromamino acid, alkane, polyethylene glycol and polypropylene glycol. Insome embodiments, amino acid is natural amino acid. In some embodiments,amino acid is unnatural amino acid. In some embodiments, an alkane is C1-C20 straight chain carbon unit. In some embodiments, polyethyleneglycol is 6 to 20 ethylene glycol unit. In some embodiments,polypropylene glycol is 6 to 20 propylene glycol unit.

In an another aspect, the present invention provides a contrast agentfor MRI having a formula selected from the group of:

In such an aspect, BP is a bisphosphonate,

is a linker, and

M is Y, In, Gd, Eu, or a lanthanide.

In one embodiment, bisphosphonate is independently selected fromalendronate, etidronate, ibandronate, incadronate, neridronate,olpadronate, phosphonate, pamidronate, risedronate, tiludronate andzoledronate. In some embodiments Eu is loaded for PARACEST contrastagent. In some embodiments, Y is loaded for hyperpolarized MRI contrastagent. In some embodiments, linker is independently selected from aminoacid, alkane, polyethylene glycol and polypropylene glycol. In someembodiments, amino acid is natural amino acid. In some embodiments,amino acid is unnatural amino acid. In some embodiments, an alkane isC1-C20 straight chain carbon unit. In some embodiments, polyethyleneglycol is 6 to 20 ethylene glycol unit. In some embodiments,polypropylene glycol is 6 to 20 propylene glycol unit.

The BP-based MRI contrast agents generated by methods of presentinvention can be used for many medical and non medical application thatwould benefit from MRI of water-poor structure such as bone lesions andtissue calcification, but none is of immediate need than breast cancerdetection. In the general population, breast cancer screening employsx-ray mammography {Van Ongeval, 2006}. In 30% to 50% of cases,microcalcification is the hallmark for the presence of cancer {Morgan,2005}, although x-ray mammography cannot distinguish the chemical formof the calcium salts present, and therefore relies on the pattern ofcrystal deposition {Stomper, 2003}. However, breast cancercalcifications are of two major types. Type I crystals, found morefrequently in benign ductal cysts, are birefringent and colorless, andare composed of calcium oxalate {Morgan, 2005}. Type II crystals, mostoften seen in proliferative lesions and associated with breast cancercells, are composed of calcium hydroxyapatite (HA), and arenon-birefringent and basophilic {Haka, 2002}. Because of the relativelylow sensitivity and specificity of x-ray mammography, MRI has become thestandard of care for screening women at high genetic risk of the disease{Saslow, 2007}. Yet, the sensitivity and specificity of MR in thissetting, estimated to be 80% and 90%, respectively {Lehman, 2007}, arestill not high enough for maximal positive- and negative-predictivevalue.

HA microcalcifications are a hallmark of malignant breast cancer butcannot be detected by current clinical MRI. The major medicalapplication of present invention is in the high sensitivity MRIdetection of tissue calcification, especially microcalcification inbreast cancer, without the need for ionizing radiation.

Present invention demonstrates an application of UTE sequences for MRIof contrast agents bound to calcifications in-vivo and in-vitro.Relaxivity properties and adsorption affinities of the complexes aretested using HA as a model of the calcification and bone surface, overother calcium salts, such as, Ca-oxalate (CO), Ca-pyrophosphate (CPP),Ca-phosphate (CP) and Ca-carbonate (CC) salts.

For in-vitro detection of HA by MRI, after a short incubation time with[Gd³⁺-DOTA]-Thr-Pam-Na (Scheme 1), UTE MRI, but not conventionalgradient echo (GRE) sequence MRI is able to detect HA crystals with highsensitivity. Signal enhancement is dependent on the concentration of[Gd³⁺-DOTA]-Thr-Pam-Na incubated with the HA crystals, with incubationconcentrations as low as 1 μM resulting in detectable signalenhancement. Signal enhancement is also dependent on relaxation time(TR), with TR≈200 msec providing the lowest background from bulk waterand the highest signal enhancement of the HA crystals.

To determine the selectivity and specificity of [Gd³⁺-DOTA]-Thr-Pam-Nafor HA, a major mineral component of calcifications and normal bone,over other calcium salts, in the present invention an incubation ofequal quantity each of Ca-hydroxyapatite (HA), Ca-pyrophosphate (CPP),Ca-phosphate (CP), Ca-oxalate (CO) and Ca-carbonate (CC) salts with[Gd³⁺-DOTA]-Thr-Pam-Na in phosphate buffered saline (PBS) is performed.UTE MRI is taken before and after washing crystals,[Gd³⁺-DOTA]-Thr-Pam-Na has more than three fold higher specificity forHA over other calcium salts found in the body, and permits MRI detectionof HA with good sensitivity.

For in-vivo detection of subcutaneously implanted HA crystals as a modelof breast cancer microcalcification, in present invention, mice withsubcutaneously implanted HA slurries (in PBS) are imaged by both UTE MRIand microCT after intravenous (IV) injection of [Gd³⁺-DOTA]-Thr-Pam-Na.After a minimum of 4 h of clearance, and consistent with the in-vitroresults, UTE MRI provides a sensitive detection of HA crystals in-vivo,with the signal enhancement corresponding to the location of the x-raydense crystals by microCT. Of note, the crystals are invisible by UTEMRI pre-injection of the contrast agent.

EXAMPLES 1. Preparation of [Gd³⁺-DOTA]-Thr-Pam-Na (Scheme 1; FIG. 7)DOTA(tBu)₃-Thr (Intermediate):

To a solution of threonine (0.19 mmol) in 0.1 mL water anddimethylformamide (DMF; 0.4 mL) at 0° C., is added triethylamine (TEA;0.38 mmol) followed by dropwise addition of DOTA(tBu)₃-NHS ester (0.12mmol) in dimethylformamide (DMF; 0.5 mL) for 10 min with stirring. After10 min, the ice bath is removed and stirring continued at roomtemperature (RT) for 16 h. The reaction mixture is poured over 2 mLice-cold water and purified by preparative HPLC.

DOTA(COOH)₃-Thr (Intermediate):

DOTA(tBu)₃-Thr (0.10 mmol) is taken in trifluoroacetic acid (TFA; 1 mL).The solution is stirred at RT for 2.5 h then the acid is removed by a N₂stream. After lyophilization, an intermediate DOTA(COOH)₃-Thr isobtained without further purification as a white powder.

[Gd³⁺-DOTA]-Thr (Intermediate):

The chelation of Gd is performed by adding 0.10 mL of 1 M GdCl₃ (0.10mmol) in water to a solution of 0.10 mmol of DOTA(COOH)₃-Thr in 0.9 mLof 0.5 M acetic acid buffer (HAc/Ac⁻), pH 5.5. The reaction mixture isstirred at RT for 12 h and purification by preparative HPLC results inan intermediate [Gd³⁺-DOTA]-Thr.

[Gd³⁺-DOTA]-Thr-Pam-Me (Intermediate):

Me-Pam {Bhushan, 2007} (0.01 mmol),O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HCTU; 0.01 mmol), and N-methylmorpholine (NMM; 0.01mmol) are added at RT under N₂ atmosphere to 0.01 mmol [Gd³⁺-DOTA]-Thrin anhydrous dimethylsulfoxide (DMSO; 0.5 mL). After stirring for 1 h atRT, the reaction mixture is poured over 3 mL ice-cold water andpurification by preparative HPLC results in an intermediate[Gd³⁺-DOTA]-Thr-Pam-Me.

[Gd³⁺-DOTA]-Thr-Pam-Na:

Trimethylsilyl bromide (Me₃SiBr; 0.04 mmol) is added slowly to asolution of [Gd³⁺-DOTA]-Thr-Pam-Me (0.01 mmol) in dry dimethylformamide(DMF; 0.1 mL) at 0° C. under nitrogen atmosphere. The reaction mixtureis vortexed at RT for 12 h. Methanolic NaOH is added to adjust pHbetween 4 and 4.2, vortexing for 30 min at RT followed by preparativeHPLC purification results in the product [Gd³⁺-DOTA]-Thr-Pam-Na.

2. Preparation of [Gd^(4′)-DOTA]-(PEG)₈-Pam-Na (Scheme 2; FIG. 8)DOTA(tBu)₄-(PEG)₈ (Intermediate):

To a solution of Amino-(PEG)₈-COOH (0.19 mmol) in 0.1 mL water anddimethylformamide (DMF; 0.4 mL) at 0° C., is added triethylamine (TEA;0.38 mmol) followed by dropwise addition of DOTA(tBu)₄-NHS ester (0.12mmol) in dimethylformamide (DMF; 0.5 mL) for 10 min with stirring. After10 min, the ice bath is removed and stirring is continued at RT for 16h. The reaction mixture is poured over 2 mL ice-cold water and anintermediate DOTA(tBu)₄-(PEG)₈ is purified by preparative HPLC.

DOTA(COOH)₄-(PEG)₈ (Intermediate):

DOTA(tBu)₄-(PEG)₈ (0.10 mmol) is taken in trifluoroacetic acid (TFA; 1mL). The solution is stirred at RT for 2.5 h then the acid is removed bya N₂ stream. After lyophilization, an intermediate DOTA(COOH)₄-(PEG)₈ isobtained without further purification as a white powder.

[Gd⁴⁺-DOTA]-(PEG)₈ (Intermediate):

The chelation of Gd is performed by adding 0.15 mL of 1 M GdCl₃ (0.10mmol) in water to a solution of 0.10 mmol of DOTA(COOH)₄-(PEG)₈ in 0.85mL of 0.5 M acetic acid buffer (HAc/Ac⁻), pH 5.5. The reaction mixtureis stirred at RT for 12 h and an intermediate [Gd⁴⁺-DOTA]-(PEG)₈ ispurified by preparative HPLC.

[Gd⁴⁺-DOTA]-(PEG)₈-Pam-Me (Intermediate):

Me-Pam (0.01 mmol),O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HCTU; 0.01 mmol), and N-methylmorpholine (NMM; 0.01mmol) are added at RT under N₂ atmosphere to 0.01 mmol[Gd⁴⁺-DOTA]-(PEG)₈ in 1 mL anhydrous dimethylsulfoxide (DMSO; 0.5 mL).After stirring for 1 h at RT, the reaction mixture is poured over 3 mLice-cold water and is purified by preparative HPLC to obtain anintermadiate [Gd⁴⁺-DOTA]-(PEG)₈-Pam-Me.

[Gd⁴⁺-DOTA]-(PEG)₈-Pam-Na:

Trimethylsilyl bromide (Me₃SiBr; 0.04 mmol) is added slowly to asolution of [Gd⁴⁺-DOTA]-(PEG)₈-Pam-Me (0.01 mmol) in drydimethylformamide (DMF; 0.1 mL) at 0° C. under nitrogen atmosphere. Thereaction mixture is vortexed at RT for 12 h. Methanolic NaOH is added toadjust pH between 4 and 4.2, being vortexed for 30 min at RT and theproduct [Gd⁴⁺-DOTA]-(PEG)₈-Pam-Na is purified by preparative HPLC.

3. Preparation of [Gd³⁺-DOTA]-Thr-Pam-Me (Scheme 3; FIG. 9)DOTA(tBu)₃-Thr-Cl (Intermediate):

DOTA(tBu)₃-Thr (0.02 mmol) is taken in tetrahydrofuran (THF; 1 mL) at 0°C. under nitrogen atmosphere, is added dimethylformamide (DMF; 5 μL) and0.04 mmol of 2 M solution of oxalyl chloride in tetrahydrofuran (THF).The solution is stirred at RT for 1 h and after that solvent is removedto get solid DOTA(tBu)₃-Thr-Cl which is used for next step reaction.

DOTA(tBu)₃-Thr-Pam-Me (Intermediate):

To DOTA(tBu)₃-Thr-Cl (0.02 mmol), is added dropwise trimethyl phosphite(0.025 mmol) at 0° C. under nitrogen atmosphere for 5 minutes and isstirred at RT for about 30 minutes. To the above reaction mixture, isadded dropwise dimethyl phosphite (0.025 mmol) at 0° C. under nitrogenatmosphere for 5 minutes and is stirred at RT for about 30 minutes thenis added 2 mL cold water and an intermediate DOTA(tBu)₃-Thr-Pam-Me ispurified by preparative HPLC.

DOTA(COOH)₃-Thr-Pam-Me (Intermediate):

DOTA(tBu)₃-Thr-Pam-Me (0.01 mmol) is taken in trifluoroacetic acid (TFA;1 mL). The solution is stirred at RT for 2.5 h then the acid is removedby a N₂ stream. After lyophilization, an intermediateDOTA(COOH)₃-Thr-Pam-Me is obtained without further purification as awhite powder.

[Gd³⁺-DOTA]-Thr-Pam-Me:

The chelation of Gd is performed by adding 0.10 mL of 1 M GdCl₃ (0.01mmol) in water to a solution of 0.01 mmol of DOTA(COOH)₃-Thr-Pam-Me in0.9 mL of 0.05 M acetic acid buffer (HAc/Ac⁻), pH 5.5. The reactionmixture is stirred at RT for 12 h and product [Gd³⁺-DOTA]-Thr-Pam-Me ispurified by preparative HPLC.

4. Preparation of [Gd⁴⁺-DOTA]-(PEG)₈-Pam-Me (Scheme 4; FIG. 10)DOTA(tBu)₄-(PEG)₈-Cl (Intermediate):

DOTA(tBu)₄-(PEG)₈ (0.02 mmol) is taken in tetrahydrofuran (THF; 1 mL) at0° C. under nitrogen atmosphere, is added dimethylformamide (DMF; 5 μL)and 0.04 mmol of 2 M solution of oxalyl chloride in tetrahydrofuran(THF). The solution is stirred at RT for 1 h and after that solvent isremoved to get solid DOTA(tBu)₄-(PEG)₈-Cl which is used for next stepreaction.

DOTA(tBu)₄-(PEG)₈-Pam-Me (Intermediate):

To DOTA(tBu)₄-(PEG)₈-Cl (0.02 mmol), is added dropwise trimethylphosphite (0.025 mmol) at 0° C. under nitrogen atmosphere for 5 minutesand is stirred at RT for about 30 minutes. To the above reaction mixtureis added dropwise dimethyl phosphite (0.025 mmol) at 0° C. undernitrogen atmosphere for 5 minutes and is stirred at RT for about 30minutes then is added 2 mL cold water and an intermediateDOTA(tBu)₄-(PEG)₈-Pam-Me is purified by preparative HPLC.

DOTA(COOH)₄-(PEG)₈-Pam-Me (Intermediate):

DOTA(tBu)₄-(PEG)₈-Pam-Me (0.01 mmol) is taken in trifluoroacetic acid(TFA; 1 mL). The solution is stirred at RT for 2.5 h then the acid isremoved by a N₂ stream. After lyophilization, an intermediateDOTA(COOH)₄-(PEG)₈-Pam-Me is obtained without further purification as awhite powder.

[Gd⁴⁺-DOTA]-(PEG)₈-Pam-Me:

The chelation of Gd is performed by adding 0.15 mL of 1 M GdCl₃ (0.01mmol) in water to a solution of 0.01 mmol of DOTA(COOH)₄-(PEG)₈-Pam-Mein 0.85 mL of 0.05 M acetic acid buffer (HAc/Ac⁻), pH 5.5. The reactionmixture is stirred at RT for 12 h and product [Gd⁴⁺-DOTA]-(PEG)₈-Pam-Meis purified by preparative HPLC.

5. Preparation of [Gd³⁺-DOTA]-Thr-Pam-Na (Scheme 5; FIG. 11)DOTA(tBu)₃-Thr-Pam/DOTA(tBu)₃-Thr-Pam-Me (Intermediate):

Pamidronic acid/Me-Pam (0.01 mmol),O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HCTU; 0.01 mmol), and N-methylmorpholine (NMM; 0.01mmol) are added at RT under N₂ atmosphere to 0.01 mmol DOTA(tBu)₃-Thr inanhydrous dimethylsulfoxide (DMSO; 0.5 mL). After stirring for 1 h atRT, the reaction mixture is poured over 3 mL ice-cold water and anintermediate DOTA(tBu)₃-Thr-Pam/DOTA(tBu)₃-Thr-Pam-Me is purified bypreparative HPLC.

DOTA (tBu)₃-Thr-Pam (Intermediate):

Trimethylsilyl bromide (Me₃SiBr; 0.04 mmol) is added slowly to asolution of DOTA(tBu)₃-Thr-Pam-Me (0.01 mmol) in dry dimethylformamide(DMF; 0.1 mL) at 0° C. under nitrogen atmosphere. The reaction mixtureis vortexed at RT for 12 h. Methanol/water (4/1) are added, beingvortexed for 30 min at RT and an intermediate DOTA(tBu)₃-Thr-Pam ispurified by preparative HPLC.

DOTA(COOH)₃-Thr-Pam (Intermediate):

DOTA(tBu)₃-Thr-Pam (0.01 mmol) is taken in trifluoroacetic acid (TFA; 1mL). The solution is stirred at RT for 2.5 h then the acid is removed bya N₂ stream. After lyophilization, an intermediate DOTA(COOH)₃-Thr-Pamis obtained without further purification as a white powder.

[Gd³⁺-DOTA]-Thr-Pam-Na:

The chelation of Gd is performed by adding 0.10 mL of 1 M GdCl₃ (0.01mmol) in water to a solution of 0.01 mmol of DOTA(COOH)₃-Thr-Pam in 0.9mL of 0.05 M acetic acid buffer (HAc/Ac), pH 5.5. The reaction mixtureis stirred at RT for 12 h and product [Gd³⁺-DOTA]-Thr-Pam-Na is purifiedby preparative HPLC.

6. Preparation of [Gd⁴⁺-DOTA]-(PEG)₈-Pam-Na (Scheme 6; FIG. 12)DOTA(tBu)₄-(PEG)₈-Pam/DOTA(tBu)₄-(PEG)₈-Pam-Me (Intermediate):

Pamidronic acid/Me-Pam (0.01 mmol),O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HCTU; 0.01 mmol), and N-methylmorpholine (NMM; 0.01mmol) are added at RT under N₂ atmosphere to 0.01 mmol DOTA(tBu)₄-(PEG)₈in anhydrous dimethylsulfoxide (DMSO; 0.5 mL). After stirring for 1 h atRT, the reaction mixture is poured over 3 mL ice-cold water and anintermediate DOTA(tBu)₄-(PEG)₈-Pam/DOTA(tBu)₄-(PEG)₈-Pam-Me is purifiedby preparative HPLC.

DOTA(tBu)₄-(PEG)₈-Pam (Intermediate):

Trimethylsilyl bromide (Me₃SiBr; 0.04 mmol) is added slowly to asolution of DOTA(tBu)₄-(PEG)₈-Pam-Me (0.01 mmol) in drydimethylformamide (DMF; 0.1 mL) at 0° C. under nitrogen atmosphere. Thereaction mixture is vortexed at RT for 12 h. Methanol/water (4/1) areadded, being vortexed for 30 min at RT and an intermediateDOTA(tBu)₄-(PEG)₈-Pam is purified by preparative HPLC.

DOTA(COOH)₄-(PEG)₈-Pam (Intermediate):

DOTA(tBu)₄-(PEG)₈-Pam (0.01 mmol) is taken in trifluoroacetic acid (TFA;1 mL). The solution is stirred at RT for 2.5 h then the acid removed bya N₂ stream. After lyophilization, an intermediateDOTA(COOH)₄-(PEG)₈-Pam is obtained without further purification as awhite powder.

[Gd⁴⁺-DOTA]-(PEG)₈-Pam-Na:

The chelation of Gd is performed by adding 0.15 mL of 1 M GdCl₃ (0.01mmol) in water to a solution of 0.01 mmol of DOTA(COOH)₄-(PEG)₈-Pam in0.85 mL of 0.05 M acetic acid buffer (HAc/Ac⁻), pH 5.5. The reactionmixture is stirred at RT for 12 h and product [Gd⁴⁺-DOTA]-(PEG)₈-Pam-Nais purified by preparative HPLC.

7. UTE MRI:

MRI can be performed on a 1.5 T GE Signa clinical scanner equipped witha custom low-pass birdcage coil (10 cm length, 6 cm diameter). Thecustom UTE sequence is based on previous work in the field {Irarrazabal,1995; Song, 1998}.

8. In-Vitro UTE and GRE MRI of HA Crystals Bound by[Gd^(3′)-DOTA]-Thr-Pam-Na:

1 mM of [Gd³⁺-DOTA]-Thr-Pam-Na is added to 5 mg of HA crystals in 50 μLPBS (pH 7.4) and is vortexed for 1 h at RT in a 1.5 mL Eppendorf tube. 5mg HA in 1 mM of [Gd³⁺-DOTA]-Thr and 50 μL PBS is used as a control.MRI, pre- and post-washing with 4×500 μL PBS, are acquired using an UTEsequence (TR=200 msec, TE=100 μsec) or conventional GRE sequence (TR=200msec, TE=1.8 msec). Other acquisition parameters includes FOV=6 cm,slice thickness=5 mm, matrix size=256×256, NEX=4.

9. Contrast Agent Concentration and TR Dependence of UTE MRI Signals:

5 mg HA is placed in 1.5 mL plastic Eppendorf tubes, then 0, 0.1, 1, 10,or 100 μM [Gd³⁺-DOTA]-Thr-Pam-Na in 50 μL PBS is added to each. Aftervortexing 1 h at RT, the crystals are washed with 4×500 μL PBS and UTEMRI acquisition is performed using a fixed TE=100 μsec and varying TR of17, 50, 200, 500 msec. Other acquisition parameters includes FOV=11 cm,slice thickness=10 mm, matrix size=256×256, NEX=2.

10. Quantitation of Calcium Salt Specificity:

5 mg of HA or the phosphate, oxalate, carbonate, or pyrophosphate saltsof calcium is placed in 1.5 ml Eppendorf tube and is incubated with 10μM [Gd³⁺-DOTA]-Thr-Pam-Na in 50 μL PBS for 1 h at RT with continuousvortexing. UTE MRI acquisition is performed pre- and post-washing with4×500 μL PBS using TR=200 msec and TE=100 μsec. Other acquisitionparameters includes FOV=9 cm, slice thickness=10 mm, matrixsize=256×256, NEX=2.

11. In-Vivo Imaging of HA:

50 mg of HA crystals is taken in 300 μL PBS, is implanted subcutaneouslyat right flank of anesthetized mice. UTE MRI is taken after implantationof HA crystal using TR/TE=200 msec/100 μsec, FOV=6 cm, slice thickness=5mm, matrix size=256×256. 4 μmol of [Gd³⁺-DOTA]-Thr-Pam-Na in 300 μLsaline is injected intravenously. After 4 h of clearance, an UTE MRI istaken with same parameters.

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What is claimed is:
 1. A contrast agent represented in general formula[I], and pharmaceutically acceptable salts, hydrates and solventsthereof:

wherein BP is a bisphosphonate;

is a linker; and

is a metal chelate selected independently from:


2. The contrast agent of claim 1, wherein said bisphosphonate isindependently selected from the group consisting of alendronate,etidronate, ibandronate, incadronate, neridronate, olpadronate,phosphonate, pamidronate, risedronate, tiludronate and zoledronate. 3.The contrast agent of claim 1, wherein said linker is selected from thegroup consisting of amino acid, alkane, polyethylene glycol andpolypropylene glycol.
 4. The contrast agent of claim 1, wherein M isselected from the group consisting of Y, In, Gd, Eu and lanthanide,wherein Gd is chelated for magnetic resonance imaging, Eu is chelatedfor CEST imaging and Y is chelated for hyperpolarized imaging.
 5. Acontrast agent represented in general formula [II], and pharmaceuticallyacceptable salts, hydrates and solvents thereof:

wherein BP is a bisphosphonate;

is a linker; and M is Y, In, Gd, Eu, or a lanthanide.
 6. The contrastagent of claim 5, wherein said linker is selected from the groupconsisting of amino acid, alkane, polyethylene glycol and polypropyleneglycol.
 7. The contrast agent of claim 5, wherein said bisphosphonate isindependently selected from the group consisting of alendronate,etidronate, ibandronate, incadronate, neridronate, olpadronate,phosphonate, pamidronate, risedronate, tiludronate and zoledronate.
 8. Acontrast agent having a formula selected from the group consisting of:

wherein BP is a bisphosphonate;

is a linker; and M is Y, In, Gd, Eu, or a lanthanide.
 9. The contrastagent of claim 8, wherein said linker is selected from the groupconsisting of amino acid, alkane, polyethylene glycol and polypropyleneglycol.
 10. The contrast agent of claim 8, wherein said contrast agentis in a form of pharmaceutically acceptable salts, hydrates andsolvents.
 11. The contrast agent of claim 8, wherein Gd is chelated formagnetic resonance imaging, Eu is chelated for CEST imaging and Y ischelated for hyperpolarized imaging.
 12. A method of making a contrastagent, said method comprising: (a) providing an organic chelatingligand, wherein said organic chelating ligand selected from the groupconsisting of:

wherein R is t-butyl ester, ester or hydrogen; and R¹ is

(b) reacting said organic chelating ligand with a linker, wherein saidlinker having a primary amine and a carboxylic moiety at opposing ends;(c) treating said carboxylic moiety with oxalyl chloride underconditions capable of forming an acid chloride at said carboxylicmoiety; (d) reacting said acid chloride in one pot with trialkylphosphite and dialkyl phosphite to form a alkylester protectedbisphosphonate; (e) deprotecting one or more carboxylic acid ester ofsaid organic chelating ligand to yield one or more carboxylic acidfunctionality; (f) chelating a metal ion to result in a metal chelate,wherein said linker separate said metal chelate and said alkylesterprotected bisphosphonate; and (g) deprotecting one or morebisphosphonate ester of said alkylester protected bisphosphonate,wherein deprotection results in said contrast agent.
 13. The method ofclaim 12, wherein said linker is selected from the group consisting ofamino acid, alkane, polyethylene glycol and polypropylene glycol. 14.The method of claim 12, wherein said metal ion is selected from thegroup consisting of Y, In, Gd, Eu and lanthanide, wherein Gd is chelatedfor magnetic resonance imaging, Eu is chelated for CEST imaging and Y ischelated for hyperpolarized imaging.
 15. A method of making a contrastagent, said method comprising: (a) providing an organic chelatingligand, wherein said organic chelating ligand selected from the groupconsisting of:

wherein R is t-butyl ester, ester or hydrogen; and R¹ is

(b) reacting said organic chelating ligand with a linker, wherein saidlinker having a primary amine and a carboxylic moiety at opposing ends;(c) deprotecting one or more carboxylic acid ester of said organicchelating ligand to yield one or more carboxylic acid functionality; (d)chelating a metal ion to result in a metal chelate, wherein said linkeron said metal chelate having said carboxylic moiety; and (e) reacting anamino bisphosphonate with said carboxylic moiety of said metal chelateunder a condition capable of forming amide bond to results in saidcontrast agent.
 16. The method of claim 15, wherein said linker isselected from the group consisting of amino acid, alkane, polyethyleneglycol and polypropylene glycol.
 17. The method of claim 15, whereinsaid amino bisphosphonate is an amino alkylester protectedbisphosphonate, wherein deprotection of one or more bisphosphonate esterresults in said contrast agent.
 18. The method of claim 15, wherein saidmetal ion is selected from the group consisting of Y, In, Gd, Eu andlanthanide, wherein Gd is chelated for magnetic resonance imaging, Eu ischelated for CEST imaging and Y is chelated for hyperpolarized imaging.19. A method of making a contrast agent, said method comprising: (a)providing an organic chelating ligand, wherein said organic chelatingligand selected from the group consisting of:

wherein R is t-butyl ester, ester or hydrogen; and R¹ is

(b) reacting an amino bisphosphonate with said organic chelating ligandunder a condition capable of forming amide bond between said aminobisphosphonate and said organic chelating ligand; (c) deprotecting oneor more carboxylic acid ester of said organic chelating ligand to yieldone or more carboxylic acid functionality; and (d) chelating a metal ionto said one or more carboxylic acid ester of said organic chelatingligand to result in said contrast agent.
 20. The method of claim 19,wherein a linker separates said organic chelating ligand and said aminobisphosphonate, wherein said amino bisphosphonate is independentlyselected from the group consisting of alendronate, neridronate, andpamidronate, wherein said linker is selected from the group consistingof amino acid, alkane, polyethylene glycol and polypropylene glycol. 21.The method of claim 19, wherein said metal ion is selected from thegroup consisting of Y, In, Gd, Eu and lanthanide, wherein Gd is chelatedfor magnetic resonance imaging, Eu is chelated for CEST imaging and Y ischelated for hyperpolarized imaging.